Recent evidence suggests that chronic infection with the respiratory pathogen, Chlamydia pneumoniae, may contribute to the development of atherosclerosis. The long range objective of this proposal is to determine the mechanisms by which infection may contribute to enhanced vascular smooth muscle cell (VSMC) proliferation, a component of early atherosclerotic lesions. The working hypothesis for these studies is that VSMC recognize and respond to cell wall components of C. pneumoniae with a strong proliferative response and upregulation of interleukin-1alpha (IL1alpha) synthesis, which in turn amplifies and sustains the initial mitogenic signal elicited by chlamydial products. Studies during the previous grant period showed that the precursor form of IL1alpha is a potent membrane-associated growth factor for human VSMC (HVSMC), and that IL1-induced proliferation involves activation of TRAF6, NIK, and IKKs, leading to the persistent activation of NF-kappaB. Preliminary data indicate that a heat-labile component of C. pneumoniae is a potent mitogenic stimulus for HVSMC. The proposed studies will determine the mechanisms of C. pneumoniae-induced HVSMC proliferation, focusing on the role of Toll-like receptor 4 (TLR4), which is expressed by HVSMC. TLRs mediate recognition of bacterial products, including lipopolysaccharide and heat shock protein 60, have intracellular domains which are homologous to the type I IL1 receptor, and likewise activate TRAF6, NIK and IKKs. Two specific hypotheses will be tested in the proposed studies. First, C. pneumoniae induces HVSMC proliferation via activation of TLR4, with subsequent recruitment of TRAF6 and ultimate activation of NF-kappaB and p44/p42 mitogen-activated protein kinases. Second, autocrine production of IL1alpha, and its myristylation-dependent localization to the cell surface, sustains and enhances the primary effects of TLR4 activation. The specific aims are: to determine whether C. pneumoniae or its molecular components induce proliferation of HVSMC via TLR4- mediated activation of TRAF6, NIK, IKK, and ultimately NF-kappaB, and by TRAF6-mediated activation of p42/p44 mitogen-activated protein kinases; to determine whether autocrine production of IL1alpha precursor contributes to the mitogenic effect of C. pneumoniae in HVSMC; and to determine whether myristylation of lysine83 plays a crucial role in the mitogenic effects of IL1alpha precursor by targeting it to the plasma membrane. The studies will employ transient transfection with dominant negative mutants, TLR4 and IL1 receptor antagonists, and antisense oligonucleotides. The results of these studies will elucidate the potential roles of bacterial and chlamydial products in the pathogenesis of vascular disease.